Induction of epithelial to mesenchymal transition in PMC42-LA human breast carcinoma cells by carcinoma-associated fibroblast secreted factors

Background Breast carcinoma is accompanied by changes in the acellular and cellular components of the microenvironment, the latter typified by a switch from fibroblasts to myofibroblasts. Methods: We utilised conditioned media cultures, Western blot analysis and immunocytochemistry to investigate the differential effects of normal mammary fibroblasts (NMFs) and mammary cancer-associated fibroblasts (CAFs) on the phenotype and behaviour of PMC42-LA breast cancer cells. NMFs were obtained from a mammary gland at reduction mammoplasty, and CAFs from a mammary carcinoma after resection. Results We found greater expression of myofibroblastic markers in CAFs than in NMFs. Medium from both CAFs and NMFs induced novel expression of α-smooth muscle actin and cytokeratin-14 in PMC42-LA organoids. However, although conditioned media from NMFs resulted in distribution of vimentin-positive cells to the periphery of PMC42-LA organoids, this was not seen with CAF-conditioned medium. Upregulation of vimentin was accompanied by a mis-localization of E-cadherin, suggesting a loss of adhesive function. This was confirmed by visualizing the change in active β-catenin, localized to the cell junctions in control cells/ cells in NMF-conditioned medium, to inactive β-catenin, localized to nuclei and cytoplasm in cells in CAF-conditioned medium. Conclusion We found no significant difference between the influences of NMFs and CAFs on PMC42-LA cell proliferation, viability, or apoptosis; significantly, we demonstrated a role for CAFs, but not for NMFs, in increasing the migratory ability of PMC42-LA cells. By concentrating NMF-conditioned media, we demonstrated the presence of factor(s) that induce epithelial-mesenchymal transition in NMF-conditioned media that are present at higher levels in CAF-conditioned media. Our in vitro results are consistent with observations in vivo showing that alterations in stroma influence the phenotype and behaviour of surrounding cells and provide evidence for a role for CAFs in stimulating cancer progression via an epithelial-mesenchymal transition. These findings have implications for our understanding of the roles of signalling between epithelial and stromal cells in the development and progression of mammary carcinoma.

Myoepithelial molecular markers in human breast carcinoma PMC42-LA cells are induced by extracellular matrix and stromal cells

The microenvironment plays a key role in the cellular differentiation of the two main cell lineages of the human breast, luminal epithelial, and myoepithelial. It is not clear, however, how the components of the microenvironment control the development of these cell lineages. To investigate how lineage development is regulated by 3-D culture and microenvironment components, we used the PMC42-LA human breast carcinoma cell line, which possesses stem cell characteristics. When cultured on a two-dimensional glass substrate, PMC42-LA cells formed a monolayer and expressed predominantly luminal epithelial markers, including cytokeratins 8, 18, and 19; E-cadherin; and sialomucin. The key myoepithelial-specific proteins α-smooth muscle actin and cytokeratin 14 were not expressed. When cultured within Engelbreth-Holm- Swarm sarcoma-derived basement membrane matrix (EHS matrix), PMC42-LA cells formed organoids in which the expression of luminal markers was reduced and the expression of other myoepithelial-specific markers (cytokeratin 17 and P-cadherin) was promoted. The presence of primary human mammary gland fibroblasts within the EHS matrix induced expression of the key myoepithelial-specific markers, α-smooth muscle actin and cytokeratin 14. Immortalized human skin fibroblasts were less effective in inducing expression of these key myoepithelial-specific markers. Confocal dual-labeling showed that individual cells expressed luminal or myoepithelial proteins, but not both. Conditioned medium from the mammary fibroblasts was equally effective in inducing myoepithelial marker expression. The results indicate that the myoepithelial lineage is promoted by the extracellular matrix, in conjunction with products secreted by breast-specific fibroblasts. Our results demonstrate a key role for the breast microenvironment in the regulation of breast lineage development.

Vibrational spectroscopy of phthalocyanine and naphthalocyanine in sandwich-type (na)phthalocyaninato and porphyrinato rare earth complexes: Part 14. The infrared and Raman characteristics of phthalocyanine in “pinwheel-like” homoleptic bis[1,8,15,22-tetrakis(3-pentyloxy)phthalocyaninato] rare earth(III) double-decker complexes

The infrared (IR) spectroscopic data and Raman spectroscopic properties for a series of 13 “pinwheel-like” homoleptic bis(phthalocyaninato) rare earth complexes M[Pc(α-OC5H11)4]2 [M = Y and Pr–Lu except Pm; H2Pc(α-OC5H11)4 = 1,8,15,22-tetrakis(3-pentyloxy)phthalocyanine] have been collected and comparatively studied. Both the IR and Raman spectra for M[Pc(α-OC5H11)4]2 are more complicated than those of homoleptic bis(phthalocyaninato) rare earth analogues, namely M(Pc)2 and M[Pc(OC8H17)8]2, but resemble (for IR) or are a bit more complicated (for Raman) than those of heteroleptic counterparts M(Pc)[Pc(α-OC5H11)4], revealing the decreased molecular symmetry of these double-decker compounds, namely S8. Except for the obvious splitting of the isoindole breathing band at 1110–1123 cm−1, the IR spectra of M[Pc(α-OC5H11)4]2 are quite similar to those of corresponding M(Pc)[Pc(α-OC5H11)4] and therefore are similarly assigned. With laser excitation at 633 nm, Raman bands derived from isoindole ring and aza stretchings in the range of 1300–1600 cm−1 are selectively intensified. The IR spectra reveal that the frequencies of pyrrole stretching and pyrrole stretching coupled with the symmetrical CH bending of –CH3 groups are sensitive to the rare earth ionic size, while the Raman technique shows that the bands due to the isoindole stretchings and the coupled pyrrole and aza stretchings are similarly affected. Nevertheless, the phthalocyanine monoanion radical Pc′− IR marker band of bis(phthalocyaninato) complexes involving the same rare earth ion is found to shift to lower energy in the order M(Pc)2 > M(Pc)[Pc(α-OC5H11)4] > M[Pc(α-OC5H11)4]2, revealing the weakened π–π interaction between the two phthalocyanine rings in the same order.

Reducing the global burden of depression : population level analysis of intervention cost-effectiveness in 14 world regions

International evidence on the cost and effects of interventions for reducing the global burden of depression remain scarce. Aims: To estimate the population-level cost-effectiveness of evidence-based depression interventions and their contribution towards reducing current burden. Method: Primary-care-based depression interventions were modelled at the level of whole populations in 14 epidemiological subregions of the world. Total population-level costs (in international dollars or I$) and effectiveness (disability adjusted life years (DALYs) averted) were combined to form average and incremental cost-effectiveness ratios. Results: Evaluated interventions have the potential to reduce the current burden of depression by 10–30%. Pharmacotherapy with older antidepressant drugs, with or without proactive collaborative care, are currently more cost-effective strategies than those using newer antidepressants, particularly in lower-income subregions. Conclusions: Even in resource-poor regions, each DALYaverted by efficient depression treatments in primary care costs less than 1 year of average per capita income, making such interventions a cost-effective use of health resources. However, current levels of burden can only be reduced significantlyif there is a substantialincrease substantial increase intreatment coverage.

Comparison between ACI 440 and FIB 14 design guidelines in using CFRP for strengthening of a concrete bridge headstock

This paper compares and reviews the recommendations and contents of the guide for the design and construction of externally bonded FRP systems for strengthening concrete structures reported by ACI committee 440 and technical report of Externally bonded FRP reinforcement for RC structures (FIB 14) in application of carbon fiber reinforced polymer (CFRP) composites in strengthening of an aging reinforced concrete headstock. The paper also discusses the background, limitations, strengthening for flexure and shear, and other related issues in use of FRP for strengthening of a typical reinforced concrete headstock structure such as durability, de-bonding, strengthening limits, fire and environmental conditions. A case study of strengthening of a bridge headstock using FRP composites is presented as a worked example in order to illustrate and compare the differences between these two design guidelines when used in conjunction with the philosophy of the Austroads (1992) bridge design code.

Comparing the ethical predisposition of university students in five English-speaking countries : an examination of 14 questionable business practices

Recent years have seen intense scrutiny focused on the reported ethical breaches of enterprises across the globe. At the forefront of the accompanying criticism are the actions of giant American firms such as WorldCom, Arthur Anderson, and Enron. However, such deviations from acceptable standards of conduct have not been confined to the American market. Australia endured its era of “corporate excess” in the 1980s [Milton-Smith, 1997]. As a result, a spate of ethics-based research was undertaken in the early 1990s. More recently, China has been identified as a major venue for behavior deemed to be unacceptable, even unsafe. Issues such as counterfeit fashion items, software, and automobile parts have been a concern for several years [Gonzalez, 2007]. Perhaps more disconcerting are the recent recalls of children’s products, many of which were produced for leading toy companies such as Mattel and Fisher-Price, because of the use of dangerous lead-based paint. As one might anticipate, news reports and consumer protection agencies have been quick to condemn any action that falls within the “controversial” category. Indeed, many segments of society characterize such actions as unethical behavior. One result of this increased level of concern is the higher level of attention given to ethics in higher education programs. Even accreditation bodies such as AACSB have virtually mandated the integration of ethics into the curriculum. As a consequence, academicians have ramped up their ethics-based research agendas.

Maternal anxiety and attention problems in children at 5 and 14 years

Objective: This study examines the association between maternal anxiety from pregnancy to 5 years and child attention problems at 5 and 14 years. Method: Birth cohort of 3,982 individuals born in Brisbane between 1981 and 1983 are assessed. Self-reported measures of maternal anxiety are assessed at four time points. Maternal reports of child attention problems using Achenbach’s Child Behavior Checklist are assessed at 5 and 14 years. Results: Children of mothers experiencing anxiety during or after pregnancy are at greater risk of experiencing attention problems at 5 and 14 years. After adjusting for maternal age and child’s gender, antenatal anxiety is strongly associated with persistent attention problems (OR = 3.65, 95% CI = 2.19, 6.07). Children with chronically anxious mothers are 5.67 (95% CI = 3.56, 9.03) times more likely to have persistent attention problems. These associations remain consistent after adjusting for potential confounders. Conclusions: Maternal anxiety appears to increase the rate of child attention problems and identifies a need for treatment programs to have a dual focus—the mother and her child. (J. of Att. Dis. 2009; XX(X) 1-XX)

Evaluation of an intervention to promote protective infant feeding practices to prevent childhood obesity : outcomes of the NOURISH RCT at 14 months of age and 6 months post the first of two intervention modules

OBJECTIVE: To evaluate a universal obesity prevention intervention, which commenced at infant age 4-6 months, using outcome data assessed 6-months after completion of the first of two intervention modules and 9 months from baseline. DESIGN: Randomised controlled trial of a community-based early feeding intervention. SUBJECTS AND METHODS: 698 first-time mothers (mean age 30±5 years) with healthy term infants (51% male) aged 4.3±1.0 months at baseline. Mothers and infants were randomly allocated to self-directed access to usual care or to attend two group education modules, each delivered over three months, that provided anticipatory guidance on early feeding practices. Outcome data reported here were assessed at infant age 13.7±1.3 months. Anthropometrics were expressed as z-scores (WHO reference). Rapid weight gain was defined as change in weight-for-age z-score (WAZ) > +0.67. Maternal feeding practices were assessed via self-administered questionnaire. RESULTS: There were no differences according to group allocation on key maternal and infant characteristics. At follow up (n=598 [86%]) the intervention group infants had lower BMIZ (0.42±0.85 vs 0.23±0.93, p=0.009) and infants in the control group were more likely to show rapid weight gain from baseline to follow up (OR=1.5 CI95%1.1-2.1, p=0.014). Mothers in the control group were more likely to report using non- responsive feeding practices that fail to respond to infant satiety cues such as encouraging eating by using food as a reward (15% vs 4%, p=0.001) or using games ( 67% vs 29%, p<0.001). CONCLUSIONS: These results provide early evidence that anticipatory guidance targeting the ‘when, what and how’ of solid feeding can be effective in changing maternal feeding practices and, at least in the short term, reducing anthropometric indicators of childhood obesity risk. Analyses of outcomes at later ages are required to determine if these promising effects can be sustained.

Non-combinatorial library screening reveals subsite cooperativity and identifies new high-efficiency substrates for kallikrein-related peptidase 14

An array of substrates link the tryptic serine protease, kallikrein-related peptidase 14 (KLK14), to physiological functions including desquamation and activation of signaling molecules associated with inflammation and cancer. Recognition of protease cleavage sequences is driven by complementarity between exposed substrate motifs and the physicochemical signature of an enzyme's active site cleft. However, conventional substrate screening methods have generated conflicting subsite profiles for KLK14. This study utilizes a recently developed screening technique, the sparse matrix library, to identify five novel high-efficiency sequences for KLK14. The optimal sequence, YASR, was cleaved with higher efficiency (k(cat)/K(m)=3.81 ± 0.4 × 10(6) M(-1) s(-1)) than favored substrates from positional scanning and phage display by 2- and 10-fold, respectively. Binding site cooperativity was prominent among preferred sequences, which enabled optimal interaction at all subsites as indicated by predictive modeling of KLK14/substrate complexes. These simulations constitute the first molecular dynamics analysis of KLK14 and offer a structural rationale for the divergent subsite preferences evident between KLK14 and closely related KLKs, KLK4 and KLK5. Collectively, these findings highlight the importance of binding site cooperativity in protease substrate recognition, which has implications for discovery of optimal substrates and engineering highly effective protease inhibitors.